Thbs1

For immunofluorescence, heart sections (8 μm) were incubated with primary antibodies against CD68 (Serotec, 1:200), CD301 (Biolegend, 1:200), PCNA (CST, 1:800), CD31 (RD, 1:200), VEGF (Proteintech, 1:200), SMA (Sigma, 1:500), CollagenΙ(Proteintech,1:200), THBS1(Proteintech, 1:200) and Alexa Fluor 488/594/633-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, 1:1,000) for 1 h at room temperature, respectively. The embedded hearts were washed and stained with DAPI (Millipore, MA). Images were captured under a Olympus (FV1000) laser-scanning confocal microscope from each heart section for further analysis. Positive staining area and relative mean fluorescent intensity were measured using Image-Pro Plus software 6.0 (Media Cybernetics, Rockville, MD, USA)50 (link).
For immunocytochemistry, primary Mos/Mps grown on slides were stimulated with or without PGE₂ and then fixed with 4% paraformaldehyde. The slides were blocked in TBST containing 1% bovine serum albumin after washing, incubated with primary antibodies against CD68 (Serotec, 1:200) or VEGF (Proteintech, 1:200) overnight at 4 °C, stained with a secondary antibody for 2 h at room temperature, and photographed using fluorescent microscopy as described above. At least five random images were taken in region of each slide and positive signalling was quantified as previously described51 (link).

The cells were seeded in a 30-mm confocal dish. After being cultured for 24 h, the dishes were fixed with 4% paraformaldehyde for 20 min and permeabilized in 0.5% Triton for 15 min. Afterward, the samples were blocked with 5% bovine serum albumin for 1 h and probed by specific primary antibodies for YTHDF1 (1:100; Proteintech, Wuhan, China), THBS1(1:100; Proteintech, Wuhan, China) at 4 °C overnight after treatment. Later they were detected with fluorescent secondary antibodies (1:100; Thermo Scientific, Wilmington, DE, USA) in a dark room, followed by 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime, Shanghai, China) for 2 min. The samples were visualized and photographed with an inverted confocal microscope (Olympus, Tokyo, Japan).